【作者】 叶蓁；

【导师】 周权；

【作者基本信息】 浙江大学 ， 药物分析学， 2010， 硕士

【摘要】 目的：1.探讨黄芪颗粒对人体P-gp活性的影响,以预期黄芪颗粒与常见合并用药相互作用的临床可能性；2.考察在中国人中MDR1 C3435T和SLCO1B1 T521C基因多态性对非索非那定(FEX)药动学的影响；3.考察黄芪颗粒对MDR1 C3435T和SLCO1B1 T521C不同基因亚组的影响。方法：1.首先建立FEX血浆浓度的测定方法。以乙腈直接沉淀法处理200μl血浆样品,高速离心后上清液吹干,复溶后直接进样分析。分析柱：Waters Symmetry C18色谱柱(4.6mm×250mm,5μm);流动相：0.1mol·L-1醋酸铵溶液-乙腈(63：37,v／v)；流速：1mL·min-1;检测器：荧光检测器(激发波长为230nm,发射波长为290nm)；以苯海拉明为内标,用内标法定量,进行色谱确证试验。2.设计一项随机、双周期、安慰剂对照临床试验,以FEX药动学参数作为人体内P-gp活性的探针。14名健康男性志愿者随机口服黄芪颗粒(4g bid)或安慰剂(4g bid),多剂量连续服用一周后,服用探针药物FEX片120mg,采集不同时间点的血浆,经色谱测定和DAS2.1.1统计软件分析,选择非房室模型计算药动学参数,并进行生物等效性统计分析。比较安慰剂组与黄芪组中FEX的药动学差异。3.利用黄芪颗粒-FEX相互作用研究中志愿者全血样本,检测MDR1 C3435T和SLCO1B1 T521C两个位点的突变情况,对不同基因型亚组中的FEX药动学参数进行数据挖掘。结果：1.本研究建立的色谱方法专属性强。在20-1000μg/L浓度范围内线性良好(r=0.9993,n=5)。定量限为20μg/L。高、中、低质控样品的日内、日间RSD均小于10%,方法学回收率为105.9%,提取回收率为94.6%。2.在安慰剂组中,FEX的药动学参数：T1/2 3.75±1.47h; Cmax 745.1±137.41μg/L; Tmax 2.25±0.47h; AUC(0-t) 3894.27±923.45μg/L*h; AUC(0-∞)3993.84±912.97μg/L*h.在黄芪颗粒组中,FEX的药动学参数：T1/2 4.00±1.24h; Cmax 709.44±170.03μg/L; Tmax 2.21±0.51h; AUC(0-t) 3832.72±1077.60μg/L*h; AUC(0-∞)3983.53±1019.83μg/L*h.黄芪颗粒对FEX的药动学影响无统计学意义(P>0.05)。FEX片在服用安慰剂组和服用黄芪颗粒组中人体内生物等效。与国外文献报道相比较,发现Cmax、Tmax和AUC0～t的差异无统计学意义(P<0.05),T1／2与国内文献报道值(5.34±1.15h)较为接近,但小于国外文献报道的9.6±2.9h。3.14名健康志愿者中MDR1 C3435T基因型的分型结果：野生型纯合子CC(n=5)；突变型纯合子TT(n=3)、突变型杂合子CT (n=6). OATP1B1 T521C基因型分型结果：TT (n=12); TC (n=2).4.MDR1 C3435T基因型与非索非那定药动学关系：突变基因T携带者中,FEX的T1／2要高于野生型CC基因型携带者中的数据(TT、CT、TT+CT> CC, P<0.01).在黄芪组中,未观察到T1／2的基因剂量效应(TT、CT、TT+CT> CC→TT、CT、TT+CT=CC, P>0.05).通过比较安慰剂组和黄芪组中相应CC基因型的药动学参数,发现T1／2的差异具有统计学意义(P<0.05,T1/2安慰剂<T1／2黄芪组)。5.FEX在安慰剂组中的PK参数并没有呈现OATP1B1 T521C的基因剂量效应。黄芪颗粒预处理后,与OATP1B1 521TT野生型纯合子相比,突变型杂合体(CT)携带者中的Cmax有升高迹象(889.50±11.54 vs 679.43±158.09μg/L, P=0.05).结论：1.本实验建立的RP-HPLC法快速简便、灵敏、专属、准确,可用于FEX的临床药动学研究；2.黄芪颗粒预处理一周不会显著影响人体P-gp活性,但它的影响具有MDR1C3435T基因型依赖性。在MDR1 C3435T基因型分亚组的情况下,显示了黄芪颗粒对P-gp的影响。黄芪颗粒对野生型等位基因个体中的P-gp有一定的抑制作用。野生型个体中的P-gp受到的抑制作用程度要大于突变型个体中P-gp受到的抑制作用程度。3.FEX消除速率可能存在民族差异性,值得进一步研究。4.黄芪颗粒预处理一周对中国健康志愿者的OATP1B1活性可能有抑制作用。更多还原

【Abstract】 Objective:1. To investigate the influences of Astragalus root extract (Huang Qi) granules on human P-gp and anticipate the risk of its drug interactions with common coadministered medications;2. To investigate the effects of MDR1 C3435T and SLCO1B1 T521C polymorphism on fexofenadine (FEX) pharmacokinetics and whether pretreatment of Huang Qi granules produces different effects on subgroup of C3435T or T521C genotype.Methods:1. An assay method was established for determining FEX concentrations in human plasma. Plasma samples(200μL) were deproteinized by precipitation with acetonitrile, centrifuged and the supernatant was reconstituted and injected into HPLC. Separation was achieved on Waters Symmetry C18 (4.6mm×250mm,5μm) with a mixture of 0.1mol/L ammonium acetate-acetonitrile (63:37, v/v) as mobile phase. The flow rate was 1mL/min. The fluorescence detector was set at excitation wavelength of 230 nm and emission wavelength of 290 nm. The internal standard method was used to quantify Fex. The assay was validated and applied to pharmacokinetic study. 2. The study had an open-label, randomized, placebo-controlled, two-period crossover design, with 3 wk washout between periods. Fex was used as an in vivo P-gp phenotyping probe.Fourteen volunteers received multiple doses of Huang Qi granules (4 g bid) or placebo (4 g bid) for one week and then received a single oral dose of 120 mg FEX tablet. Plasma samples were taken at different times and analysed by chromatography. Pharmacokinetic parameters were calculated by non-compartmental method and compared in two groups. Bioequivalence evaluation was performed by the software DAS2.1.1.3. C3435T mutation in exon 26 (MDR1 C3435T) and SLCO1B1 T521C mutation were determined in this pharmacokinetic study. Data mining was performed.Results:1. With respect to chromatographic analysis, endogenous chemicals did not interfere with Fex. The calibration curves were linear in the range of 20～1000μg/L (r=0.9993, n=5), with a limit of quantitation of 20μg/L. The within-and between-day RSDs of quality-control samples at high-, medium-and low-concentrations were less than 10%. The average method recovery was 105.9%. The average absolute recovery was 94.6%.2. Pharamcokinetic parameters derived from placebo group were as follows:T1/2(3.75±1.47 h); Cmax (745.11±137.41μg/L); Tmax (2.25±0.47 h); AUC(0-t) (3894.27±923.45μg/L*h); AUC(0-∞) (3993.84±912.97μg/L*h).Pharamcokinetic parameters derived from Huang Qi group were as follows::T1/2(4.00±1.24 h); Cmax(709.44±170.03μg/L); Tmax (2.21±0.51 h); AUC(0-t) (3832.72±1077.60μg/L*h); AUC(0-∞) (3983.53±1019.83μg/L*h). The effects of Hunag Qi granules on FEX pharmcokinetics were insignificant (P>0.05). FEX tablets in two groups were bioequivalent. The average elimination half life was similar with data derived from Liu’s study in 20 Chinese volunteers, but it was shorter than documented data (9.6±2.9 h) abroad. With respect to Cmax, Tmax and AUCo-t, no significant differences were obversed(P<0.05).3. MDR1 C3435T phenotyping results of 14 healthy volunteers were as follows: hemozygous CC (wide type, n=5); hemozygous TT(mutation type,n=3); heterozygous CT(mutation type,n=6). OATP1B1 T521C phenotyping results were as follows:TT(n=12); TC (n=2).4. In MDR1 3435 T allele carriers, T1/2 of FEX was longer than that in MDR1 3435 CC carriers (TT, CT, TT+CT> CC, P<0.01). Gene-dose effects on T1/2 in Huang Qi group were not observed (TT, CT, TT+CT> CC→TT, CT, TT+CT=CC, P> 0.05). By comparison of pharmacokinetic parameters in MDR1 3435 CC carriers in either placebo group or Huang Qi group, difference in T1/2 was significant(P=0.02, T1/2(Placebo)< T1/2(Huangqi)).5. There were no gene-dose effects of OATP1B1 521 C on pharmacokinetic parameters of fexofenadine in placebo group. Cmax in OATPIB1 521 C allele carriers exhibited an increase trend with marginal significance (889.50±11.54 vs 679.43±158.09μg/L, P=0.05) compared with that in OATP1B1 521 TT carriers.Conclusion:1. The RP-HPLC assay is stable, simple, rapid, accurate, sensitive and applicable for determining plasma concentrations of FEX in its pharmacokinetic studies;2. One-week pretreatment of Huang Qi granules has no significant effects on human P-gp activity in Chinese healthy subjects. However, effect of Huang Qi granules on P-gp was MDR1 C3435T genotype-dependent. Huang Qi granules may have inhibitory effects on P-gp activity in MDR1 3435 CC carriers. The decrement degree in 3435 CC carriers was greater than that in 3435 T allele carriers.3. The terminal elimination profile of FEX indicates ethnic differences and further investigation is needed.4. One-week pretreatment of Huang Qi granules may have inhibitory effects on OATP1B1 activity in Chinese healthy subjects.更多还原